The DEAD-box ATPase Dbp10/DDX54 initiates peptidyl transferase center formation during 60S ribosome biogenesis

Cruz, Victor E.; Weirich, Christine S.; Peddada, Nagesh; Erzberger, Jan P.

doi:10.1038/s41467-024-47616-7

Download PDF

Article
Open access
Published: 17 April 2024

The DEAD-box ATPase Dbp10/DDX54 initiates peptidyl transferase center formation during 60S ribosome biogenesis

Nature Communications volume 15, Article number: 3296 (2024) Cite this article

807 Accesses
1 Altmetric
Metrics details

Subjects

Abstract

DEAD-box ATPases play crucial roles in guiding rRNA restructuring events during the biogenesis of large (60S) ribosomal subunits, but their precise molecular functions are currently unknown. In this study, we present cryo-EM reconstructions of nucleolar pre-60S intermediates that reveal an unexpected, alternate secondary structure within the nascent peptidyl-transferase-center (PTC). Our analysis of three sequential nucleolar pre-60S intermediates reveals that the DEAD-box ATPase Dbp10/DDX54 remodels this alternate base pairing and enables the formation of the rRNA junction that anchors the mature form of the universally conserved PTC A-loop. Post-catalysis, Dbp10 captures rRNA helix H61, initiating the concerted exchange of biogenesis factors during late nucleolar 60S maturation. Our findings show that Dbp10 activity is essential for the formation of the ribosome active site and reveal how this function is integrated with subsequent assembly steps to drive the biogenesis of the large ribosomal subunit.

The RNA helicase Dbp7 promotes domain V/VI compaction and stabilization of inter-domain interactions during early 60S assembly

Article Open access 22 October 2021

Association of snR190 snoRNA chaperone with early pre-60S particles is regulated by the RNA helicase Dbp7 in yeast

Article Open access 22 October 2021

Sequence-specific remodeling of a topologically complex RNP substrate by Spb4

Article 08 December 2022

Introduction

Accurate assembly of large (60S) and small (40S) ribosomal subunits requires an elaborate, multicompartmental biogenesis pathway^1,2. During this maturation process, primary rRNA transcripts undergo co-transcriptional modifications and nucleolytic processing before folding into a compact tertiary structure stabilized by interactions with ribosomal proteins (RPs). The initial, spontaneous folding of primary rRNA transcripts favors the formation of local stem loops, which must then assemble into correct helical junctions using long-range secondary and tertiary interactions to form larger rRNA domains^3,4 (Supplementary Fig. 1a). RPs guide and accelerate this process, stabilizing unique tertiary structures to define the ribosome assembly pathway. In eukaryotes, this process is further facilitated by more than 200 ribosome biogenesis factors (RBFs)^5,6 that transiently engage pre-ribosomes in a coordinated, hierarchical manner to enable the high rates of ribosome production required for cellular growth and division⁷.

One crucial rRNA element of the 60S is the universally conserved peptidyl transferase center (PTC), which brings together A- and P-site tRNAs and catalyzes peptide bond formation. Within the PTC lies the A-loop, which caps helix H92 and orients the CCA tail of the A-site tRNA, ensuring the proper orientation of the acceptor amino acid within the PTC⁸. The position of the A-loop within the PTC is defined by an intricate three-way junction that anchors rRNA helices H90, H91 and H92 and is only peripherally bound by RPs⁹. To date, there is no structural information on how these tertiary rRNA interactions form during 60S assembly.

PTC assembly and/or quality control has been proposed to require the activity of DEAD-box ATPases, ubiquitous RNA-directed enzymes with wide-ranging functions throughout RNA biology^{10,11,12,13,14}. In prokaryotes, the DEAD box protein DbpA directly associates with H92 via a C-terminal RNA-binding domain^15,16. While there is no eukaryotic homolog of DbpA, Dbp10/DDX54, another DEAD-box ATPase, has been linked to rRNA helices H90-H91-H92 through CRAC analysis¹⁷. More recently, cross-linking mass spectrometry and structural studies have placed Dbp10/DDX54 on the nascent subunit interface, flanked by the RBFs Nop2, Nog1 and Nsa2^18,19,20, but these studies have not defined the catalytic function of Dbp10/DDX54 or revealed how the early PTC is formed.

In this study, we set out to investigate the possible role of Dbp10 in PTC formation during nucleolar pre-60S assembly. Using a slow-hydrolysis mutant, we pinpoint the precise timing of Dbp10 catalytic function during 60S maturation and present three cryo-electron microscopy (cryo-EM) reconstructions of pre-catalytic, catalytic and post-catalytic pre-60S states that establish the function of Dbp10 in PTC formation. The pre-catalytic structure reveals an unexpected, alternate secondary structure of helix H92 prior to Dbp10 engagement. Unwinding and remodeling of this rRNA structure by Dbp10 forms the A-loop and facilitates the assembly of the H90/H91/H92 three-way junction at the core of the PTC. Finally, the post-catalytic Dbp10-pre60S complex communicates these structural changes in the PTC to other regions of the pre-60S, coupling the formation of the ribosome active site to the compositional re-structuring of the pre-60S particle.

Results

Genetic and structural characterization of Dbp10 function

Because Dbp10 has been purified as part of several pre-60S particles during nucleolar 60S maturation, we first set out to define the precise timing of Dbp10 catalytic rRNA engagement. The active site architecture at the interface of the two RecA domains (D1 and D2) that form the enzymatic core of DEAD-box ATPases is highly conserved, allowing us to use insights from previous studies^21,22,23 to generate Dbp10 mutants that fail to bind ATP and substrate (Dbp10^K187A, Dbp10^K187R, and Dbp10^D288A) or mutants that can bind ATP and ssRNA, but are deficient in hydrolysis (Dbp10^E289A and Dbp10^R522V) (Fig. 1a). Genetically, these slow-hydrolysis mutants are predicted to become kinetically trapped on the pre-60S substrate and therefore display a dominant negative phenotype in vivo.

**Fig. 1: Defining the timing of Dbp10 catalytic activity during 60S assembly.**

In a DBP10 shuffle strain, dbp10^K187A, dbp10^K187R and dbp10^D288A all fail to rescue the lethality of a dbp10∆ allele, while mutants dbp10^E289A and dbp10^R522V display a slow growth phenotype (Fig. 1b). Consistent with our hypothesis, estradiol-inducible overexpression of dbp10^R522V results in a dominant negative phenotype (Fig. 1c), while no effect is observed upon overexpression of variants mutated in motifs I (dbp10^K187A) or II (dbp10^D288A) (Fig. 1c), likely because these mutants never engage the pre-60S. The growth defect of the dbp10^R522V strain is due to a 60S maturation defect, as determined by polysome profiling of dbp10^R522V extracts (Fig. 1d). Monitoring of ribosome biogenesis in vivo using GFP-tagged large (uL23-GFP) or small (uS7-GFP) subunits shows that the observed decrease in mature 60S subunits is due to nuclear/nucleolar accumulation of pre-60S subunits (Fig. 1e). To identify the pre-60S substrate for Dbp10, we overexpressed DBP10 or dbp10^R522V and purified pre-60S intermediates with split tags on Dbp10 and the RBF Brx1, which is present on pre-60S intermediates before and after Dbp10 binding. Semi-quantitative mass-spectrometry analysis of these purified intermediates reveals that, compared with overexpression of DBP10, overexpression of dbp10^R522V leads to an increase in the abundance of Rrp14, Rrp15, and Ssf1 and a decrease in the levels of Noc2, Noc3, Spb1, and Nug1 (Fig. 1f). Our results are broadly consistent with a study of Dbp10 mutants published while this manuscript was under review²⁴. This factor exchange also coincides with the docking of rRNA domain III to the pre-60S core, indicating that Dbp10 remains bound during this major assembly event (Fig. 2a). Thus Dbp10, like the DEAD-box ATPases Has1 and Spb4, remains bound to the pre-60S post-catalytically, a state recently captured in cryo-EM reconstructions of human and C. thermophilum pre-60S intermediates^19,20. These studies also show that another RBF exchange, the removal of the Nsa1/Mak16/Rpf1/Rrs1 module by Rix7, appears to occur independently from Dbp10 function.

**Fig. 2: Cryo-EM reconstructions of Dbp10 pre-catalytic, catalytic, and post-catalytic intermediates.**

We next set out to isolate pre-60S intermediates representing pre-catalytic, catalytic and post-catalytic states of Dbp10, using distinct genetic and biochemical trapping strategies (Fig. 2a and Supplementary Fig. 1b, c): Purification of pre-60S particles from a strain overexpressing dbp10^R522V with split-tags on Dbp10^R522V and Ssf1 yielded purified particles with stoichiometric amounts of Dbp10. However, cryo-EM reconstructions of this sample yielded a reconstruction that lacked any density for Dbp10, likely because it dissociated during grid preparation. A skip align 3D-classification focused on the region predicted by XL-MS to represent the binding region of Dbp10¹⁸ revealed a subset of particles with a distinct, previously uncharacterized structural feature. Because Dbp10^R522V is catalytically impaired, we reasoned that the resulting 2.5 Å reconstruction might represent the pre-catalytic state. (Fig. 2b, Supplementary Figs. 2, 3). We confirmed this by reconstructing the structure of pre-60S particles from a strain overexpressing a slow-hydrolysis mutant of a different DEAD-box ATPase, Drs1(Cruz, et al., unpublished data), which binds Ssf1-containing pre-60S particles immediately before Dbp10^18,25,26. Pre-60S particles isolated from this strain lack Dbp10 (Supplementary Fig. 1d) but retain the same PTC architecture as the reconstruction from the dbp10^R522V overexpression strain (Cruz, et al., unpublished data and Supplementary Fig. 1e).

A 2.7 Å reconstruction of the catalytic Dbp10 intermediate was obtained from pre-60S intermediates purified from wild-type Dbp10 steady state cells using split affinity tags on Dbp10 and Ssf1, stabilized by the addition of the γ-phosphate mimic beryllium fluoride (BeF₃) and low concentrations of the bifunctional crosslinker DSS (Supplementary Figs. 1, 4 and 5). BeF₃ allows DEAD box ATPases to unwind dsRNA and subsequently stabilizes a DEAD-box-BeF₃-ssRNA (i.e. substrate bound) complex^23,27,28,29. This structure is compositionally identical to the pre-catalytic state, except for the presence of Dbp10 (Fig. 2b, c and Supplementary Fig. 1b, c).

Finally, the post-catalytic state was obtained from a strain harboring a genomic 3´ auxin-inducible degron (AID) fused to the DEAD-box ATPase SPB4. Addition of 3-indole acetic acid (auxin) to these strains led to a significant enrichment of the Dbp10 post-catalytic state (Supplementary Fig. 1c), purified using split affinity tags on Dbp10 and Noc2. A single particle reconstruction from this sample yielded a 2.7 Å reconstruction (Fig. 2d, Supplementary Figs. 6 and 7). The molecular composition of these three intermediates is consistent with our mass-spectrometry analysis, confirming the concerted exchange of essential RBFs during late nucleolar assembly while Dbp10 remains engaged on the pre-60S: The pre-catalytic and catalytic structures are characterized by the presence of Rrp14, Rrp15, and Ssf1, while in the post-catalytic state, these factors are replaced by Noc2, Noc3 and Spb1 (Fig. 2 and Supplementary Fig. 1b, c).

Structure of the immature PTC

Our reconstruction of the pre-60S prior to Dbp10 catalysis captures the PTC immediately after the initial docking of three of its constituent rRNA helices (H89, H91 and H93) to the pre-60S core (Fig. 3a). This initial step is mediated by a set of RBFs that individually stabilize these helices: Nsa2 and Nog1 engage both H89 and H91 (Fig. 3b, c), while Nip7 binds the H93 stem-loop (Fig. 3d). In previously characterized structures of pre-60S intermediates, the elements connecting these helices could not be resolved and were assumed to be too dynamic to be captured by cryo-EM^20,30. Our improved reconstruction now shows that, unexpectedly, the H92 stem and the A-loop do not form co-transcriptionally, but instead fold into an alternate base-pairing arrangement (Fig. 3e, f). In our model, four A-loop nucleotides (G2922-C2925) are base paired with a complementary sequence (G2939-C2942) that is part of H90 in the mature ribosome (Fig. 3g). Rather than forming the compact RNA junction observed in the mature ribosome (Fig. 3h, i), the immature architecture of H90/H92 extends away from the pre-60S surface and is stabilized by the RP uL14, which engages two separate regions within the alternate H91/H90/H92 structure (Fig. 3g).

**Fig. 3: Organization and structure of the Dbp10 substrate.**

Formation of a compact H90/H91/H92 junction is essential for A-loop stabilization of the aminoacylated CCA tail of the A-site tRNA (Fig. 3h). Therefore, the immature conformation we observe in the pre-catalysis intermediate must be restructured to form the functional PTC (Fig. 3i). Because RBF interactions with flanking rRNA helices (H89, H91 and H92) topologically restrict the pre-PTC scaffold, any RNA unwinding activity that locally destabilizes the alternate H90/H92 duplex will introduce a localized topological strain to facilitate the transition to the mature PTC secondary structure. Furthermore, since the number of base-pairs in the two conformations is comparable, the transition between the two secondary structures could be achieved merely by lowering the thermodynamic barrier between the two structures (Fig. 3f, h).

Dbp10 unwinding activity destabilizes the immature A-loop

To stabilize the catalytic state of Dbp10, we purified steady-state intermediates from DBP10 cells in the presence of beryllium fluoride (BeF₃) to stabilize substrate-bound intermediates. The RBF composition of this intermediate is indistinguishable from the pre-catalysis state, except for extra density corresponding to the C-terminal portion of Dbp10 in the cleft between Nop2 and Nip7 on one side and Nsa2, Nog1 and uL14 on the other (Fig. 4a). We were able to build an atomic model for the D2 domain of Dbp10 as well as additional C-terminal elements into this map (Fig. 4b, c). The main 60S anchoring interaction of Dbp10 is mediated by a series of helices within a conserved C-terminal extension (CTE) (Fig. 4b–d). The first three CTE helices wrap around the D2 domain in a triangular arrangement, mediating interactions with uL14, Nog1 and Nsa2, while an extended helical finger, composed of an additional pair of helices, points away from the D2 domain, making extensive interactions with Nog1, the N-terminal helix of Nsa2 and with rRNA helices H43 and H44 (Fig. 4d). Beyond the CTE, the C-terminal tail (CTT) of Dbp10 is entirely disordered in this reconstruction, except for a 16 amino acid segment (residues 781–796), that interacts with the RBF Nop2 (Fig. 4b–d). We tested the importance of these structural elements in vivo using a DBP10 shuffle strain. Consistent with its pre-60S anchoring function, removal of the helical finger element (dbp10^∆667-731) results in a lethal phenotype (Fig. 4e). In contrast, alanine substitutions within the Nop2-interaction motif (dbp10^∆Nop2int) results in a temperature-sensitive phenotype at 37 °C (Fig. 4f), suggesting that the Nop2 interaction is not required for initial binding, but may instead help stabilize the mobile RNA segment termed the L1 stalk (helices 75–78), especially at higher temperatures.

**Fig. 4: Structural analysis of catalytic Dbp10•pre-60S intermediates.**

Previous structural and biochemical studies of DEAD-box dsRNA unwinding using the model protein Mss116 established different functions for the two core domains: The D1 domain binds ATP while the D2 domain binds one of the strands of the duplex substrate (Fig. 4g)^28,31,32. Cooperative assembly of a bipartite interface then leads to strand unwinding and stabilization of a closed D1/D2 interface containing ATP and ssRNA^28,31,33. We reasoned that if the alternate H90/H92 structure is the Dbp10 substrate, then it must be properly oriented for recognition by the D2 domain. To determine the relative positions of D2 in the catalytic state and H92alt in the pre-catalytic state, we aligned the two models using uL14 as a guide. Superposition of H92alt onto the catalytic model (Fig. 4h and Supplementary Movie 1) shows that the duplex region formed by A-loop nucleotides is in the proper orientation to be engaged by the D2 domain, requiring only a ~4 Å rigid body swivel to place duplexed A-loop residues onto the dsRNA-recognition surface of the D2 domain of Dbp10 (Fig. 4i).

We observed no defined density for the D1 domain and the rRNA sequence spanning H90/H92 in our initial maps (Fig. 4j). We hypothesized that our BeF₃ trapping strategy may only have succeeded for a subset of particles; to isolate this subset we performed two rounds of skip-align local classification with masks centered on density observed in low-pass filtered maps near the expected position of the D1 domain (Supplementary Fig. 4). This strategy allowed us to identify a particle subset with additional density adjacent to the D2 domain (Fig. 4j). The local resolution of this density is ~6 Å, allowing us to manually dock a model for the Dbp10 D1 domain and rigid-body refine it against a map low-pass filtered to 6 Å (Fig. 4k).

A comparison of the resulting D1/D2 interface with a model of ssRNA-substrate bound Mss116 shows that the D1 model is in an orientation that closely matches the substrate-bound D1/D2 interface (Fig. 4j), although the rigid-body refined D1-D2 interface is not fully engaged and we observe no clear density for bound ssRNA. The absence of defined substrate ssRNA at the D1/D2 interface could be due either to the presence of multiple states representing intermediates between H92 alt and the mature PTC conformation or because of destabilization of the complex during grid preparation. In particular, because the DSS crosslinker added to stabilize our intermediate can readily form monolinks with lysine residues, we speculate that the Dbp10/ssRNA complex formed in the presence of ADP-BeF₃ may be disrupted and that the local map therefore represents an average of multiple orientations of the D1/D2 interface. The relative positions of the D1 and D2 domains are nevertheless consistent with our proposed model: The D2 domain is in the proper position to bind the alternate H90/H92 duplex region, and subsequent co-operative assembly of the D1/D2 interface unwinds the duplex and captures A-loop residues in the active site (Fig. 4m). This unwinding activity directly destabilizes the alternate structure of H90/H92 and favors the formation of the mature PTC secondary structure, since capture of A-loop residues in the Dbp10 active site does not interfere with the formation of the mature H90 and H92 stems.

The CTT stabilizes the Dbp10 post-catalytic state

Our model of the Dbp10 post-catalytic state reflects the extensive RBF exchange that occurs after H90/H92 remodeling by Dbp10 (Fig. 2a, b). In this intermediate, Ssf1, Rrp14 and Rrp15 have been released from the pre-60S and replaced with a new set of RBFs; the Noc2/Noc3 heterodimeric complex bridges the L1 stalk and the newly docked rRNA domain III, stabilized by the extensive interaction network of the hub RBF Spb1^34,35(Figs. 2e and 5a and Supplementary Fig. 1a). In this intermediate, the C-terminal portion of Dbp10 remains bound to the same pocket observed in the catalytic state, and in contrast with the catalytic state, the D1 domain is now clearly defined, allowing a complete atomic model of the catalytic domains of Dbp10 to be built (Fig. 5b). As expected, the H90 and H92 stems have their mature secondary structure and the compact tertiary junction anchoring H92 and the A-loop has also adopted its mature structure (Fig. 5c). This transition is consistent with our model, in which ATP hydrolysis and substrate release allow for the mature PTC structure to form (Fig. 5d).

**Fig. 5: The post-catalytic Dbp10-pre60S structure.**

Substrate release by Dbp10 does not lead to its disengagement from the pre-60S. Instead, three structural modules within the CTT interact with RBFs and rRNA elements to form a stable, non-catalytic assembly of the Dbp10 D1/D2 interface, in which the D1 domain is translated by ~10 Å and rotated by ~5° compared to the orientation of the catalytic intermediate (Fig. 6b). The first element (residues 808–834) runs across the subunit interface, directly stabilizing the non-catalytic D1/D2 interface and occupying the ssRNA binding pocket (Fig. 6b, c). The second element (residues 857–882) docks onto the D1 domain and mediates interactions with the upper segment of rRNA helix H61 (Fig. 6c). A third, more distal element (residues 918–946) runs along H64 and forms a specific interaction with the RBF Rlp24 (Fig. 6c). The H61/H64 junction, rather than being the substrate for Dbp10, is therefore bound post-catalytically. These C-terminal features are conserved in other post-catalysis structures of Dbp10/DDX54 (Supplementary Fig. 8a, b).

**Fig. 6: Dbp10 promotes RBF exchange post-catalsis.**

To understand the importance of the post-catalytic structure of Dbp10, we tested CTT deletions and alanine mutagenesis in the DBP10 shuffle strain (Fig. 6a, d). Removal of the entire CTT tail resulted in a non-viable phenotype, even when a nucleolar targeting sequence (NoLS) was added to replace the predicted localization signal at the extreme C-terminus of Dbp10 (Fig. 6a, d). In contrast to the tail deletions, mutating or removing individual structural modules had no effect on growth (Fig. 6a, d), indicating that under normal growth conditions, disruption of individual contacts between the Dbp10 CTT and the pre-60S are not essential and suggesting a degree of redundancy between individual elements in stabilizing the post-catalytic Dbp10 intermediate.

Helix H61 capture couples Dbp10 function to RBF exchange

Out of the six domains that make up the large ribosomal subunit, domain IV is the last one to become organized. In the pre-catalytic and catalytic states, the only ordered domain IV element is rRNA helix H61, which connects domain IV to the rest of the 25S rRNA. This conformation of H61, which is ~40 Å from the RNA binding pocket of Dbp10, is maintained by a conserved contact with the RBF Rrp14 (Fig. 6e). In the post-catalysis state, Rrp14 is no longer bound and the position of H61 pivots by ~60° about its anchoring point, allowing the H61/H64 junction to be engaged by Dbp10. Capture of the H61/H64 junction by Dbp10 disrupts the Rrp14/H61 interaction, weakening the affinity of Rrp14 for the pre-60S and promoting its dissociation (along with Ssf1 and Rrp15) from the pre-60S. Loss of Rrp14 exposes a core rRNA element formed by rRNA helices H25A and H32 which in the post-catalytic structure is occupied by the essential C-terminal module of Spb1 (Fig. 6f). Spb1 is a long, modular protein that acts as a hub protein to organize multiple RBFs during late nucleolar maturation^34,35. Together, these sequential steps establish a chronology of how the remodeling function of Dbp10 is directly coupled to the changes in RBF composition during late nucleolar 60S maturation.

Discussion

During RNP assembly, RNA folds in two phases: the initial co-transcriptional folding of stem-loop structures followed by the slower assembly of helical junctions and long-distance interactions to stabilize tertiary structures. During ribosome assembly, the latter is facilitated by the hierarchical incorporation of ribosomal proteins that stabilize these interactions. In bacteria, initial PTC formation appears to follow this pattern; stem-loops form their mature secondary structures prior to their full assembly into the PTC³⁶. Surprisingly, our structural data reveal that, in eukaryotes, the universally conserved stem-loop H92 initially forms a different secondary structure and must be actively restructured before the tertiary structure of the PTC core can form. Restructuring is carried out by Dbp10/DDX54, which unwinds the immature H90/H92 duplex in an ATP-dependent manner to catalyze the formation of the mature PTC tertiary structure. One consequence of this mechanism is that additional sequence restraints are imposed on the PTC rRNA, to satisfy two sets of compatible base-pairing interactions. Consistent with this, the degree of domain-specific conservation within the PTC is significantly higher in eukaryotes compared to archaea and bacteria^37,38. A direct sequence comparison between S.cerevisiae and H.sapiens PTC sequences shows that all nucleotides required for the alternate H92 base pairing are identical (Supplementary Fig. 8c, d), implying a conserved function for Dbp10/DDX54 in eukaryotes. In contrast, Dbp10 function is distinct from the bacterial DEAD-box ATPase DbpA, which has a specific recognition domain that binds the mature form of the A-loop^39,40 and therefore does not appear to be involved in promoting H92 formation. The topology of the Dbp10 substrate is also important. The localized strain imposed by the unwinding of the alternate conformation of H92 is only effective in inducing the restructuring of the alternate conformation because flanking PTC stem-loops are bound by RBFs. Local topology is also important for the function of the DEAD-box ATPase Spb4³⁴. Our structures and proposed remodeling mechanism are therefore consistent with the localized duplex-unwinding model of DEAD-box ATPase function⁴¹ and with the local topology constraints of the rRNA substrate⁴². Because Dbp10 is essential for H92 formation, our model also explains why Dbp10 function is necessary for A-loop methylation by the RBF Sbp1, as only mature A-loops are recognized by this enzyme^24,34,35.

In addition to its essential catalytic role in PTC assembly, Dbp10 also plays a structural, post-catalytic role in guiding the ordered exchange of RBFs in late nucleolar 60S assembly. Upon substrate release, the core domains of Dbp10 form a non-catalytic interface that captures the H61/H64 rRNA junction, a transition directly stabilized by several structural elements within the Dbp10 CTT. This transition communicates the successful completion of PTC assembly to more distal regions of the pre-60S by facilitating the release of Rrp14 and its interaction partners Ssf1 and Rrp15. In turn, Rrp14 release removes a steric block to allow the organization of the C-terminal anchoring domain of Spb1. In addition to its C-terminal methyltransferase domain, Spb1 contains multiple modules that interact with 15 other pre-60S components, including the Noc2/Noc3 heterodimer, acting as a critical hub to assemble rRNA domain III^{19,20,30,34,35}. These sequential events therefore directly link PTC maturation and domain III organization, revealing how a non-equilibrium, ATP-dependent step is directly connected to subsequent structural changes in the pre-60S. After rRNA domain III is assembled, the pre-60S is acted on by another DEAD-box ATPase, Spb4, which catalyzes the organization of the H61/H62/H63/H64 junction, directly linking sequential ATP-dependent steps^34,35.

The recent publication of cryo-EM structures of human and C. thermophilum pre-60S intermediates bound by Dbp10/DDX54 in their post-catalytic configuration allow for a comparison of these steps across several eukaryotic species^19,20. In both studies, intermediates were captured by pulling-down RBFs present throughout early and late assembly steps, capturing additional post-catalysis Dbp10/DDX54 intermediates that we did not observe with our SPB4 depletion strategy and providing additional details on the sequential organization of rRNA domain IV. Despite the overall structural similarities across the two species, a comparison of these intermediates also suggests some variability in the order in which Rrp14/Rrp15/Ssf1 release and Noc2/Noc3/Spb1 binding occurs. This flexibility may also explain the high degree of functional redundancy observed in our mutational analysis of post-catalytic S.cerevisiae Dbp10 CTT elements. Our data and these studies are consistent with a model in which enzymatic steps such as those catalyzed by Dbp10/DDX54 require a highly defined substrate and are therefore highly conserved across all eukaryotes, while the order of non-energy consuming binding and release events is less rigidly constrained, allowing organism-specific variability.

The catalytic function of Dbp10 shows how limited local strand unwinding can trigger the remodeling of topologically restricted rRNA sequences. We had previously proposed a similar model for the DEAD-box ATPase Spb4, and it seems likely that at least a subset of the remaining 16 structurally uncharacterized DEAD-box ATPases involved in ribosome biogenesis function in a similar manner. Dbp10 and Spb4 both contribute to the formation of complex three-way rRNA junctions. Efficient formation of these rRNA junctions is important for two reasons. The first relates to the incredible demands placed on ribosome production in fast growing cells. For example, in yeast, approximately 2000 ribosomes per minute must be synthesized to maintain exponential growth, a rate requiring energy-consuming enzymes to accelerate kinetically slow RNA folding steps¹⁴. The second relates to the quality control mechanisms that ensure misfolded ribosomal intermediates are rapidly identified and recycled. While the precise details of this process are not understood, kinetic proofreading has emerged as the leading hypothesis, proposing that failure to precisely execute rate-limiting steps during RNP biogenesis results in dead-end, degradation-prone intermediates^43,44. This latter function may explain why DEAD-box ATPases are retained post-catalysis. Dbp10 is the third structurally characterized eukaryotic DEAD-box ATPase, after Has1 and Spb4, that remains associated with the pre-60S post-catalytically, suggesting that it is a common feature in eukaryotic ribosome assembly and may be a mechanism to communicate quality control during ribosome biogenesis. Finally, because Spb4 unwinds a segment of H63, the stabilization of the immature H61/H64 junction by Dbp10 opens the intriguing possibility that the post-catalysis structure of Dbp10 facilitates initial substrate binding by Spb4, thereby directly coupling sequential DEAD-box protein unwinding events during ribosome biogenesis.

Methods

Yeast strains

Yeast strains used in this study are listed in Supplementary Table 1, plasmids used in this study are listed in Supplementary Table 2. Genomic knockout strains in yeast were performed using standard PCR protocols. For overexpression of DBP10 variants, strains were grown overnight in YPD and then serially diluted onto either YPD or YPD + 2 µM β-estradiol and grown at 30 °C for 2 days. For shuffle assays, shuffle strains were transformed with the indicated plasmid, re-streaked onto selective media (SCD -LEU or SCD-HIS), and then grown overnight in selective media and serially diluted onto either selective media or 5-FOA and grown for 3 days at 30 °C.

Plasmid construction

DBP10 centromeric plasmids were generated as follows. A genomic fragment spanning 247 bp upstream DBP10 to 66 bp downstream was cloned into pBluescript (Stratagene) using SacI/XhoI. The same sequence was cloned into pRS316 to generate pJE728 and pRS315 to generate pJE729. Point mutations and internal deletions were introduced into pJE729 using Quikchange (Agilent) to generate plasmids pJE748, pJE749, pJE750, pJE751, pJE753, pJE788, and pJE792. A synthesized DNA fragment (Genscript) was introduced carrying silent mutations in amino acids T188 and A189 to introduce an EagI site, V327 and D328 to introduce a SalI site, V565 and D566 to remove a SalI site, and amino acids L821, N822 and S823 to introduce an EcoRI site. The SacI/XhoI fragment was cloned into pRS313 using SacI and EcoRI to generate pJE1218. To generate pJE1222 (pRS313-dbp10^∆nop2int), a synthesized DNA fragment spanning BamHI to EcoRI was introduced to mutate the sequence from amino acids 787 to 794 from TFFLSHY to AAAASSA. To generate plasmid pJE1388, a synthesized DNA fragment was subcloned into pJE1218 deleting amino acids 926–943. To generate pJE1390, a synthesized DNA fragment was subcloned into pJE1322 mutating amino acids 931–934 from KHRD to AAAA. Plasmids pJE1384, pJE1386 and pJE1388 were generated using Quikchange and pJE1218 as a template. DBP10 variants were introduced into overexpression plasmid pJE664 using XhoI/AscI.

Microscopy

Strains were grown overnight in synthetic complete -uracil (SC-URA) medium with 2% dextrose, then diluted to an OD600 of 0.1 in SC-URA + 2% dextrose and grown to an OD of 0.4–0.5. Images were taken on a Nikon EclipseTi inverted microscope equipped 100× NA1.45 objective and an Andor Zyla 5.5 sCMOS camera. Image processing was performed using ImageJ.

Polysome profiling

Cells were grown in YPD to mid-log phase (optical density at 600 nm (OD600) ≈ 0.8,) then treated with 50 µg/ml of cycloheximide for 5 min prior to centrifugation and snap-freezing. Thawed cells were washed and resuspended in lysis buffer (200 mM Tris-KCl, pH 7.4, 500 mM KCl, 100 mM MgCl₂, 1 mM DTT). Extracts were prepared by glass bead lysis (8 × 30 s) and clarified by centrifuging at 4 °C for 15 min at 11,300 × g. Then, 10 OD260 units of clarified extract were loaded onto 5–50% sucrose gradients made in the same buffer. Gradients were centrifuged for 2.5 h at 260,000 × g in a Beckman SW41 rotor and RNA content was monitored at 254 nm using a Gradient Master (BioComp).

Overexpression and purification of Dbp10-containing pre-ribosomes

For yeast overexpression, full length wild-type or R522V catalytic mutant of DBP10 was cloned into a modified pRS406 plasmid that introduced a N-terminal 3xStrep-sfGFP-bdNEDD-MYC tag under the control of a β-estradiol inducible system. As a second affinity purification tag, BRX1, SSF1 or NOC2 was tagged at the genomic locus with a C-terminal 3xFLAG–3C–2xProteinA. Starter cultures for each strain were grown overnight in YPD to saturation. The following morning 12 L of YPD were inoculated from the starters to an OD600 of 0.1 and grown at 30 °C to an OD600 of 0.8. Overexpression was induced for 1 h by the addition of β -estradiol to a final concentration of 2 µM. Cells were harvested by centrifugation for 20 min at 4000 × g, the pellets were washed in Ribo-buffer A (50 mM Bis-Tris-Propane HCl pH 8.0, 125 mM NaCl, 25 mM KCl, 10 mM MgCl2, 1 mM TCEP and 0.1% (w/v) NP-40) and centrifuged again for 10 min at 4000 × g. Cell pellets were harvested then forced through a syringe into liquid nitrogen resulting in flash frozen yeast noodles. Cryogenic lysis was done using a grinding ball mill (Fritsch Pulverisette 6). For purification of pre-ribosomes, 30 g of lysate are warmed to 4 °C before addition of Ribo-buffer A supplemented with E64, pepstatin, PMSF and RNAse-free DNase I. Lysate was cleared by centrifuging at 100,000 × g for 30 min and loaded onto IgG Sepharose resin (Cytiva). Samples were washed with 10 column volumes of Ribo-buffer A followed by 10 column volumes of Ribo-buffer B (50 mM Bis-Tris-Propane HCl pH 8.0, 125 mM NaCl, 25 mM KCl, 10 mM MgCl2, 1 mM TCEP and 0.01% (w/v) NP-40). Protein-A tags were cleaved on-column for 1 h using 200 µg of 3C protease diluted into 3 mL of Ribo-buffer B and eluates were applied to a Strep-Tactin column (Cytiva), washed with 5 column volumes of Ribo-buffer B and also eluted by 30 min of on-column cleavage using 1 mL Ribo-buffer B supplemented with 50 µg of bdNEDD8 protease. The second elution for steady state sample derived from YJE1243 was instead performed with Ribo-buffer B supplemented with 10 mM desthiobiotin. In either case, eluted pre-ribosomes were concentrated on Amicon Ultra 0.5 ml spin columns with a 100KDa cutoff (Merck Millipore). Purification of post-catalysis Dbp10 which required depletion of SPB4 was performed by adding 3-Indole Acetic acid (IAA) to a final concentration of 0.5 mM at mid-log phase (OD600 of 0.8) for 45 min prior to harvesting, subsequent purification steps were identical to steady-state samples described above. For catalytic Dbp10·Ssf1 (steady state) and post-catalytic Dbp10∙Noc2 (SPB4-AID) purification, all buffers were supplemented with 50 µM ADP-BeF3.

Cryo-EM grid preparation and data collection

Cryo-EM grids were prepared by applying 3.5 µL of pre-ribosome sample at a concentration of 5 A260ml-1 to glow-discharged continuous carbon coated Quantifoil R2/1 300-mesh grids. Grids were blotted and frozen in liquid ethane using a Mark IV Vitrobot (FEI) set at 4 °C and 100% humidity. Micrographs were acquired on a Titan Krios (FEI) operated at 300KV equipped with a Gatan K3 direct electron detector using a slit width of 30 eV on a GIF-Quantum energy filter. Automated data collection was performed using SerialEM⁴⁵ with a defocus range of −0.9 to −2.2 µm and a pixel size of 1.08 Å. Each micrograph was dose fractionated into 30 frames of 0.05 s each under a dose rate of 26.2 e⁻/pixel/s with a total exposure time of 1.5 s and a dosage of approximately 39.3 e⁻/pixel. 10,879 movies were collected for the pre-catalytic sample from the inducible dominant-negative strain, 20,892 movies for the catalytic, steady state sample and 9452 movies for the post-catalytic sample from the SPB4-AID strain. The pre-catalytic sample was collected at the PNCC using a Titan Krios operated at 300KV with a pixel size of 1.0688 Å using the same parameters and defocus range as the other samples.

Cryo-EM image processing

Motion correction was performed using MotionCorr2⁴⁶, and CTF parameters were estimated using GCTF⁴⁷. All subsequent image processing was performed using RELION4⁴⁸.

Pre-catalytic Dbp10^R522V∙Ssf1 dataset

About 300 particles were picked to generate initial 2D classes to serve as templates for automated particle picking from the 10,539 micrographs. 2734K particles were extracted, binned 4 times, and used for 2D classification, from these, 2374K particles were selected for 3D classification. All identical pre-60S classes were combined and re-extracted at the original pixel size of 1.08 Å, resulting in the total of 1863K particles. CTF and 3D refinement was performed with an imposed C1 symmetry and resulted in a reconstruction with a resolution of 2.66 Å without postprocessing. Three rounds of local skip-align 3D classification (T-factors of 60, 60 and 40) were performed using a mask surrounding the PTC region to subtract core density. From this, 375K particles were selected for the final reconstruction to an overall resolution of 2.52 Å. Additional local refinements were performed to improve the density around the L1 stalk, the PTC region and the Rrp14/Rrp15/Ssf1 region to facilitate model building in these regions. The final resolution in each case was estimated by applying a soft mask and was calculated using the gold-standard Fourier shell correlation (FSC) = 0.143⁴⁹. Local resolution for all maps were generated using the ResMap⁵⁰ wrapper within Relion4.

Catalytic Dbp10∙Ssf1 (steady state) dataset

Initial 2D classes and processing was performed identically to the pre-catalytic dataset. Data from two separate grids (identical sample) were combined: in total, 3849K particles were extracted after automated particle picking from 20054 micrographs, binned 4 times and used for 2D classification. 2094K particles were selected for subsequent 3D classification. Identical classes were combined and re-extracted at the original pixel size of 1.08 Å, resulting in a combined total of 889K particles. 3D refinement was performed with an imposed symmetry of C1 and resulted in a reconstruction with a resolution of 2.9 Å before post-processing. Two rounds of local skip-align 3D classification (T-factors of 60 and 40) were performed using a mask surrounding the Dbp10 D1 region to subtract core density. From this, 195K particles were selected for the final reconstruction to an overall resolution of 2.7 Å. A local refinement of the domain VI region was also performed to improve the maps for model building and rigid body refinement. The final resolution in each case was estimated by applying a soft mask and was calculated using the gold-standard Fourier shell correlation (FSC) = 0.143⁴⁹. Local resolution for all maps were generated using the ResMap⁵⁰ wrapper within Relion4.

Post-catalytic Dbp10∙Noc2 (SPB4-AID) dataset

Initial data processing was performed as described for the Dbp10∙Ssf1 (steady-state) dataset. 1202K particles were selected from 8882 micrographs, binned 4 times and extracted for 2D classification. 869K particles were selected for 3D classification. Identical classes were combined resulting in 491K particles selected for further processing. Two rounds of local skip-align 3D classification (T-factors of 60 and 40) was performed using a mask surrounding the Dbp10/H64/H61region to subtract core density, yielding a final subset of 197K particles. After CTF refinement, polishing and postprocessing, a final map with a resolution of 2.66 Å was obtained. A local refinement of the domain VI region was also performed to improve the maps for model building. Two improve the maps surrounding the Dbp10-D1/H61/H64 region, another round of skip align classification (T-factor = 40) was performed with a more focused mask. Local refinement of this subset yielded improved maps of the H61/H64 region for model building.

Cryo-EM model building and refinement

PDB 6EM1 was used as a starting model for the pre-catalytic Dbp10^R522V∙Ssf1 intermediates and the refined pre-catalytic model was used for building and refining into the catalytic Dbp10∙Ssf1 (steady state) map. The C-terminal RecA domain of Dbp10, along with the CTE and CTT were built de novo into this map. For the post-catalytic Dbp10∙Noc2 (SPB4-AID) dataset, we used PDB 7R7A as a starting model and built the Dbp10 D1 domain using the Alphafold model⁵¹. All models were built in Coot⁵² and refined in PHENIX using phenix.real_space_refine⁵³. Figures were generated with UCSF Chimera X⁵⁴, movies were generated using PyMOL Molecular Graphics System (Version 2.0 Schödinger, LLC.).

Purification of Brx1-Dbp10 containing pre-ribosomes

Starters of strains harboring a genomic C-terminal 3xFLAG–3C–2xProteinA tag on BRX1 and either an inducible DBP10 or dbp10^R522V were grown overnight in YPD to saturation. The following morning 12 L of YPD were inoculated from the starters to an OD600 of 0.2 and grown at 30 °C to an OD600 of 0.8 and overexpression was induced by adding β-estradiol to a final concentration of 2 µm. Cells were harvested and lysed identically to samples used for cryo-EM, described above. Lysate was cleared in the same way and loaded onto an IgG Sepharose resin (Cytiva). Samples were washed with 10 column volumes of Ribo-buffer A followed by 10 column volumes of Ribo-buffer B. Protein-A tags were cleaved on-column using 3C protease and eluates were directly loaded onto a Strep-Tactin column (Cytiva), washed with 5 column volumes of Ribo-buffer B and eluted by 30 min of on-column cleavage using 1 mL Ribo-buffer B supplemented with 50 µg of bdNEDD8 protease. Samples were concentrated to an A260ml⁻¹ of 3 (100 nM) using an Amicon Ultra 0.5 ml spin column with a 100KDa cutoff (Merck Millipore) to an OD260 absorbance of 5.0.

Semi-quantitative mass-spectrometry analysis

10 µL of Dbp10 or Dbp10^R522V pre-ribosomes at an OD260 absorbance of 5.0 were run on a 4–20% denaturing Gel (BioRad) at 160 V for 7 min and stained with Coomassie Brilliant Blue R-250 (Thermo Fisher). The portion of the gel with protein bands was excised and used for subsequent analysis. Semi-quantitative mass-spectrometry data was obtained by the UTSW Proteomics core, analyzed using Proteome Discoverer 2.4 and searched against the yeast protein database from UniProt. Only high-confidence spectra (FDR ≤ 1%) were considered and are listed in Source Data. Selection of RBFs for our plot was based on their presence in known intermediate structures or well-established presences in specific pre-60S intermediates.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The cryo-EM density maps and models generated in this study have been deposited in the EMDB and PDB databases under accession codes: EMD-43017/PDB-8V83 (pre-catalytic structure – overall map and model), EMD-43018 (pre-catalytic structure – Local map PTC focused), EMD-43019 (pre-catalytic structure – Local map L1 focused), EMD-43020 (pre-catalytic structure – Local map Rrp14/Rrp15/Ssf1 focused), EMD-43021/PDB-8V84 (catalytic structure – overall map and model), EMD-43022 (catalytic structure – Local map Dbp10 focused), EMD-43024 (catalytic structure – Local map L1 focused), EMD-43026 (catalytic structure – Local map Rrp14/Rrp15/Ssf1 focused), EMD-43023/PDB-8V85 (catalytic structure – 6 Å low-pass-filtered, locally-refined map and docked D1 model), EMD-43027/PDB-8V87 (post-catalytic structure – overall map and model), EMD-43028 (post-catalytic structure – Local map Dbp10 focused), EMD-43029 (post-catalytic structure – Local map H61/H64 focused). Strain and plasmid information, cryo-EM micrographs, 2D classes, FSC curves, ResMap plots, and data processing 3D-classification and particle sorting schemes generated in this study are provided in the Supplementary Information. Source data are provided with this paper.

References

Kressler, D., Hurt, E. & Baßler, J. A Puzzle of Life: Crafting Ribosomal Subunits. Trends Biochem.Sci. 42, 640–654 (2017).
Article CAS PubMed Google Scholar
Klinge, S. & Woolford, J. L. Ribosome assembly coming into focus. Nat. Rev. Mol. Cell Biol. 20, 116–131 (2018).
Woodson, S. A. RNA folding pathways and the self-assembly of ribosomes. Acc. Chem. Res. 44, 1312–1319 (2011).
Article CAS PubMed PubMed Central Google Scholar
Rodgers, M. L. & Woodson, S. A. A roadmap for rRNA folding and assembly during transcription. Trends Biochem. Sci. 46, 889–901 (2021).
Article CAS PubMed PubMed Central Google Scholar
Woolford, J. L. & Baserga, S. J. Ribosome biogenesis in the yeast Saccharomyces cerevisiae. Genetics 195, 643–681 (2013).
Article CAS PubMed PubMed Central Google Scholar
Dorner, K., Ruggeri, C., Zemp, I. & Kutay, U. Ribosome biogenesis factors-from names to functions. EMBO J. 42, e112699 (2023).
Article CAS PubMed PubMed Central Google Scholar
Warner, J. R. The economics of ribosome biosynthesis in yeast. Trends Biochem. Sci. 24, 437–440 (1999).
Article CAS PubMed Google Scholar
Schmeing, T. M., Huang, K. S., Strobel, S. A. & Steitz, T. A. An induced-fit mechanism to promote peptide bond formation and exclude hydrolysis of peptidyl-tRNA. Nature 438, 520–524 (2005).
Article ADS CAS PubMed Google Scholar
Buschauer, R. et al. The Ccr4-Not complex monitors the translating ribosome for codon optimality. Science 368, eaay6912 (2020).
Article CAS PubMed PubMed Central Google Scholar
Martin, R., Straub, A. U., Doebele, C. & Bohnsack, M. T. DExD/H-box RNA helicases in ribosome biogenesis. RNA Biol. 10, 4–18 (2013).
Article CAS PubMed PubMed Central Google Scholar
Rodriguez-Galan, O., Garcia-Gomez, J. J. & de la Cruz, J. Yeast and human RNA helicases involved in ribosome biogenesis: current status and perspectives. Biochim. Biophys. Acta 1829, 775–790 (2013).
Article CAS PubMed Google Scholar
Jarmoskaite, I. & Russell, R. RNA helicase proteins as chaperones and remodelers. Annu Rev. Biochem. 83, 697–725 (2014).
Article CAS PubMed PubMed Central Google Scholar
Karbstein, K. Attacking a DEAD problem: The role of DEAD-box ATPases in ribosome assembly and beyond. Methods Enzymol. 673, 19–38 (2022).
Article CAS PubMed PubMed Central Google Scholar
Bohnsack, K. E., Yi, S., Venus, S., Jankowsky, E. & Bohnsack, M. T. Cellular functions of eukaryotic RNA helicases and their links to human diseases. Nat. Rev. Mol. Cell Biol. 24, 749–769 (2023).
Article CAS PubMed Google Scholar
Diges, C. M. & Uhlenbeck, O. C. Escherichia coli DbpA is an RNA helicase that requires hairpin 92 of 23S rRNA. EMBO J. 20, 5503–5512 (2001).
Article CAS PubMed PubMed Central Google Scholar
Wurm, J. P., Glowacz, K. A. & Sprangers, R. Structural basis for the activation of the DEAD-box RNA helicase DbpA by the nascent ribosome. Proc. Natl Acad. Sci. USA 118, e2105961118 (2021).
Article CAS PubMed PubMed Central Google Scholar
Manikas, R.-G., Thomson, E., Thoms, M., Hurt, E. & The, K. -dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation. Nucleic Acids Res. 44, 1800–1812 (2016).
Article PubMed PubMed Central Google Scholar
Sailer, C. et al. A comprehensive landscape of 60S ribosome biogenesis factors. Cell Rep. 38, 110353 (2022).
Article CAS PubMed PubMed Central Google Scholar
Vanden Broeck, A. & Klinge, S. Principles of human pre-60S biogenesis. Science 381, eadh3892 (2023).
Article CAS PubMed Google Scholar
Lau, B. et al. Mechanism of 5S RNP recruitment and helicase-surveilled rRNA maturation during pre-60S biogenesis. EMBO Rep. 24, e56910 (2023).
Article CAS PubMed Google Scholar
Pause, A. & Sonenberg, N. Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J. 11, 2643–2654 (1992).
Article CAS PubMed PubMed Central Google Scholar
Liu, F., Putnam, A. & Jankowsky, E. ATP hydrolysis is required for DEAD-box protein recycling but not for duplex unwinding. PNAS 51, 20209–20214 (2008).
Article Google Scholar
Liu, F., Putnam, A. A. & Jankowsky, E. DEAD-box helicases form nucleotide-dependent, long-lived complexes with RNA. Biochemistry 53, 423–433 (2014).
Article CAS PubMed Google Scholar
Mitterer, V. et al. The RNA helicase Dbp10 coordinates assembly factor association with PTC maturation during ribosome biogenesis. Nucleic Acids Res. 52, 1975–1987 (2024).
Saveanu, C. et al. Sequential Protein Association with Nascent 60S Ribosomal Particles. Mol. Cell. Biol. 23, 4449–4460 (2003).
Article CAS PubMed PubMed Central Google Scholar
Sanghai, Z. A. et al. Modular assembly of the nucleolar pre-60S ribosomal subunit. Nature 556, 126–129 (2018).
Article ADS CAS PubMed PubMed Central Google Scholar
Montpetit, B. et al. A conserved mechanism of DEAD-box ATPase activation by nucleoporins and InsP6 in mRNA export. Nature 472, 238–242 (2011).
Article ADS CAS PubMed PubMed Central Google Scholar
Mallam, A. L., Sidote, D. J. & Lambowitz, A. M. Molecular insights into RNA and DNA helicase evolution from the determinants of specificity for a DEAD-box RNA helicase. Elife 3, e04630 (2014).
Article PubMed PubMed Central Google Scholar
Ngo, T. D., Partin, A. C. & Nam, Y. RNA Specificity and Autoregulation of DDX17, a Modulator of MicroRNA Biogenesis. Cell Rep. 29, 4024–4035.e4025 (2019).
Article CAS PubMed PubMed Central Google Scholar
Kater, L. et al. Visualizing the Assembly Pathway of Nucleolar Pre- 60S Ribosomes. Cell 171, 1599–1610.e1513 (2017).
Article CAS PubMed PubMed Central Google Scholar
Mallam, A. L. et al. Solution structures of DEAD-box RNA chaperones reveal conformational changes and nucleic acid tethering by a basic tail. Proc. Natl Acad. Sci. USA 108, 12254–12259 (2011).
Article ADS CAS PubMed PubMed Central Google Scholar
Mallam, A. L., Del Campo, M., Gilman, B., Sidote, D. J. & Lambowitz, A. M. Structural basis for RNA-duplex recognition and unwinding by the DEAD-box helicase Mss116p. Nature 490, 121–125 (2012).
Article ADS CAS PubMed PubMed Central Google Scholar
Samatanga, B. & Klostermeier, D. DEAD-box RNA helicase domains exhibit a continuum between complete functional independence and high thermodynamic coupling in nucleotide and RNA duplex recognition. Nucleic Acids Res. 42, 10644–10654 (2014).
Article CAS PubMed PubMed Central Google Scholar
Cruz, V. E. et al. Sequence-specific remodeling of a topologically complex RNP substrate by Spb4. Nat. Struct. Mol. Biol. 29, 1228–1238 (2022).
Article CAS PubMed PubMed Central Google Scholar
Mitterer, V. et al. Concurrent remodelling of nucleolar 60S subunit precursors by the Rea1 ATPase and Spb4 RNA helicase. Elife 12, e84877 (2023).
Article CAS PubMed PubMed Central Google Scholar
Davis, J. H. et al. Modular Assembly of the Bacterial Large Ribosomal Subunit. Cell 167, 1610–1622.e1615 (2016).
Article CAS PubMed PubMed Central Google Scholar
Doris, S. M. et al. Universal and domain-specific sequences in 23S-28S ribosomal RNA identified by computational phylogenetics. RNA 21, 1719–1730 (2015).
Article CAS PubMed PubMed Central Google Scholar
Prosdocimi, F. et al. The Ancient History of Peptidyl Transferase Center Formation as Told by Conservation and Information Analyses. Life 10, 134 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Hardin, J. W., Hu, Y. X. & McKay, D. B. Structure of the RNA binding domain of a DEAD-box helicase bound to its ribosomal RNA target reveals a novel mode of recognition by an RNA recognition motif. J. Mol. Biol. 402, 412–427 (2010).
Article CAS PubMed PubMed Central Google Scholar
Wurm, J. P. Structural basis for RNA-duplex unwinding by the DEAD-box helicase DbpA. RNA 29, 1339–1354 (2023).
Article CAS PubMed PubMed Central Google Scholar
Russell, R., Jarmoskaite, I. & Lambowitz, A. M. Toward a molecular understanding of RNA remodeling by DEAD-box proteins. RNA Biol. 10, 44–55 (2014).
Article Google Scholar
Bailor, M. H., Mustoe, A. M., Brooks, C. L. 3rd & Al-Hashimi, H. M. Topological constraints: using RNA secondary structure to model 3D conformation, folding pathways, and dynamic adaptation. Curr. Opin. Struct. Biol. 21, 296–305 (2011).
Article CAS PubMed PubMed Central Google Scholar
Houseley, J. & Tollervey, D. The many pathways of RNA degradation. Cell 136, 763–776 (2009).
Article CAS PubMed Google Scholar
Parker, M. D. & Karbstein, K. Quality control ensures fidelity in ribosome assembly and cellular health. J. Cell Biol. 222, e202209115 (2023).
Article CAS PubMed PubMed Central Google Scholar
Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).
Article PubMed Google Scholar
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Article CAS PubMed PubMed Central Google Scholar
Zhang, K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).
Article ADS CAS PubMed PubMed Central Google Scholar
Scheres, S. H. W. Processing of Structurally Heterogeneous Cryo-EM Data in RELION. 1 ed., 579 (Elsevier Inc., 2016).
Scheres, S. H. W. & Chen, S. Prevention of overfitting in cryo-EM structure determination. Nat. Methods 9, 853–854 (2012).
Article CAS PubMed PubMed Central Google Scholar
Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2013).
Article PubMed PubMed Central Google Scholar
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
Article ADS CAS PubMed PubMed Central Google Scholar
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Cryst. D60, 2126–2132 (2004). 1-7.
CAS Google Scholar
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Cryst. D66, 213–221 (2010).
Google Scholar
Pettersen, E. F. et al. UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).
Article CAS PubMed Google Scholar

Download references

Acknowledgements

We thank Sahana Balasubramanian and Rafal Piwowarczyk for generating strains and plasmids during the early stages of this project. We also would like to thank the UTSW core facilities that have supported this work: Daniel Stoddard and the staff the UTSW Cryo-Electron Microscopy Facility, funded in part by the CPRIT Core Facility Support Award RP170644, James Chen at the UTSW Structural Biology Lab, Andrew Lemoff at the UTSW Proteomics Core and Marcel Mettlen at the UTSW Quantitative Light Microscopy Core, a shared resource of the Harold C. Simmons Cancer Center, supported in part by an NCI Cancer Center Support Grant, 1P30 CA142543-01. A portion of this research was supported by NIH grant U24GM129547 and performed at the PNCC at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research, with the assistance of Theo Humphreys. J.P.E. was supported by the Cancer Prevention and Research Institute of Texas (RR150074), the Welch Foundation (I-1897), the UTSW Endowed Scholars Fund and the National Institutes of Health (GM135617-01).

Author information

Victor E. Cruz
Present address: O’Donnell Brain Institute/CAND, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX, 75390, USA
Nagesh Peddada
Present address: Center for the Genetics of Host Defense, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX, 75390, USA

Authors and Affiliations

Department of Biophysics, UT Southwestern Medical Center - ND10.124B, 5323 Harry Hines Blvd., Dallas, TX, 75390, USA
Victor E. Cruz, Christine S. Weirich, Nagesh Peddada & Jan P. Erzberger

Authors

Victor E. Cruz
View author publications
You can also search for this author in PubMed Google Scholar
Christine S. Weirich
View author publications
You can also search for this author in PubMed Google Scholar
Nagesh Peddada
View author publications
You can also search for this author in PubMed Google Scholar
Jan P. Erzberger
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

V.E.C., C.S.W, N.P and J.P.E. conceived the project. V.E.C., C.S.W., and N.P. constructed strains. V.E.C., and N.P. carried out sample preparations and collected cryo-EM data. C.S.W. conducted polysome profiling, growth, and microscopy assays. V.E.C., and J.P.E. processed the cryo-EM data. The paper was written by J.P.E., V.E.C. and C.S.W and edited by V.E.C., C.S.W. and J.P.E. with input from N.P. J.P.E. supervised the work.

Corresponding author

Correspondence to Jan P. Erzberger.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Peer Review File

Description of Additional Supplementary Files

Supplementary Movie 1

Reporting Summary

Source data

Source Data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Cruz, V.E., Weirich, C.S., Peddada, N. et al. The DEAD-box ATPase Dbp10/DDX54 initiates peptidyl transferase center formation during 60S ribosome biogenesis. Nat Commun 15, 3296 (2024). https://doi.org/10.1038/s41467-024-47616-7

Download citation

Received: 12 December 2023
Accepted: 04 April 2024
Published: 17 April 2024
DOI: https://doi.org/10.1038/s41467-024-47616-7

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.